Hexa- and heptapeptides related to the c-terminal sequence of eledoisin



United States Patent HEXA- AND HEPTAPEPTIDES RELATED TO THE C-TERMINALSEQUENCE OF ELEDOISIN Klaus Liibke, Eberhard Schriider, Reinhard Hempel,and Ralph Schmiechen, Berlin, Germany, assignors to ScheringAktiengesellschaft, Berlin, Germany No Drawing. Filed Feb. 27, 1964,Ser. No. 347,950 Claims priority, application Germany, Mar. 1, 1963, Sch32,860; Oct. 31, 1963, Sch 34,086; Jan. 4, 1964, Sch 34,464

13 Claims. (Cl. 260-1125) The present invention relates to newpolypeptides and to methods of producing the same, and more particularlyto heptapeptides and hexapeptides.

It is an object of the present invention to provide new synthetichexapeptides and heptapeptides.

It is another object of the present invention to provide methods ofproducing the new polypeptides, e.g. hexapeptides and heptapeptides ofthe present invention.

It is another object of the present invention to provide newhexapeptides and heptapeptides which have .a highly effective bloodpressure lowering action.

It is yet another object of the present invention to provide for thereduction of the blood pressure of patients requiring the same by theadministration of the new hexapeptides and heptapeptides of thisinvention.

Other objects and advantages of the present invention will be apparentfrom a further reading of the specification and of the appended claims.

With the above and other objects in view, the present invention mainlycomprises a polypeptide of the formula:

R-L-alanyl-L-phenylalanyl L oz aminoacyl-glycyl L-leucyl-L-methioninamide wherein R is either hydrogen or a naturalLot-amino acid radical other than L-aspartyl, and wherein L-a-aminoacylis selected from the group consisting of L-isoleucyl, L- leucyl andL-valyl radicals.

The natural L-a-amino acid radicals of the substituent R are preferablythe L-forms of the radicals of glycine, alanine, valine, leucine,isoleucine, asparagine, glutamine, serine, threonine, cysteine,methionine, phenylalanine, tyrosine, proline, oxyproline, lysine,histidine, arginine, ornithine, norleucine, norvaline, a-aminobutyricacid, a,'y-diaminobutyric acid, pyroglutamic acid, the N-methylaminoacids such as sarcosine, N-methylvaline, N-methyl leucine, andtryptophane.

It is known that there is present in the salivary glands of marineanimals which manufacture and can discharge inks, such as sepia,cuttlefish, squid and the octopus, for example eledone moschata, containthe undecapeptide eledoisin of the formula:

L-pyroglutamyl-L-prolyl-L-sery1-L-lysyl L aspara gyl- L alanyl Lphenylalanyl L isoleucyl glycyl L- methioninamide which has a bloodpressure lowering action, a vessel dilating action, and which also actsto stimulate breathing.

This substance consequently is a valuable therapeutic substance.

It has been found according to the present invention that thehexapeptides and heptapeptides of the general formula:

R-L-alanyl-L-phenylalanyl L-a aminoacyl glycyl L-leucyl-L-methioninamide wherein R and L-a-aminoacyl have the samedefinitions as above, quite surprisingly has the same or practically thesame activity in the lowering of blood pressure, blood vessel dilationand stimulation of breathing as does eledoisin. In addition, these newhexapeptides and heptapep tides of the present invention provideadvantages as compared to eledoisin.

The exchange of amino acids in the 1-position of the above generalformula of the new polypeptides of the present invention, and thealternate exchange of isoleucine, valine and leucine in the 4-positionare independent of each other in their influence on the activity of thepolypeptides of the present invention, and also, independent of theexchange in the other positions of the molecule. It has been found thatthe action of the three analogs with isoleucine, valine and leucine isin the ratio of 10: 10: 1.

The use of hexapeptides instead of the undecapeptide for the samepurposes already constitutes a considerable advantage because of thesmaller molecule size, and in addition, the further advantage isobtained in connection with the arbitrary incorporation of naturalL-a-amino acids in the l-position of the heptapeptides. These advantagesare of a therapeutic nature and also in connection with the manufactureof the final preparations.

From the standpoint of manufacture of the final preparations, it isadvantageous to use in the l-position of the polypeptides of the presentinvention simple amino acids such as glycine, alanine, valine andleucine. By doing this it is possible to avoid the disadvantages of thecorresponding use of complicated amino acids, such advantages as sidereactions and lower yields.

It is further advantageous to use such amino acids which increase thewater solubility of the final product, for example lysine, histidine,arginine, threonine, serine or tyrosine. The increased solubilitysimplifies not only the synthesis itself, but also the purification ofthe final product.

Furthermore, the increased water solubility constitutes :a therapeuticadvantage since it makes possible to administer higher doses inrelatively smaller amounts of liquids. This is particularly advantageousin the case of subcutaneous administration.

The limiting dose at which the polypeptides of the present invention arestill active is, in the case of subcutaneous administration, about ofthe limiting dose in the case of intravenous administration. (Forcomparison: In the case of eledoisin, according to V. Ersparmer and A.Glaesser, Brit. J. of Pharmacol. and Chemotherapy, 20, 516 (1963) ondogs the limiting dose intravenously is 02-5 ng./kg., whilesubcutaneously it is l-Zy/kg.) To dissolve a therapeutically sufi'icientamount of, for example, I.-lysyl-L-alanyl-L-phenyl-alanyl-L-valyl-glycyl L leucyl-L-methioninamide, it is sutlicient to use 0.2-0.5 cc. of water perrabbit, while, for example, to dissolve an amount of equal activity ofLasparagylor L-asparaginyl-L-leucyl-L-alanyl-L-phenylalanyl-L-isoleucyl-glycyl L methioninamide orL-glycyl-L alanyl-L-phenylalanyl-Lvalylglycyl-L-leucyl-L-methioninamide, about 5 cc. of water is needed.Solutions of the last named compound, because of the requirement of toogreat a volume of water, there fore are not particularly suitable forsubcutaneous administration.

The particular advantage of subcutaneous administration is in the factthat the duration of the blood pressure lowering is increased about tenfold thereby.

The following table gives the blood pressure lowering effect on rabbitsupon intravenous administration of various doses of the polypeptides ofthe present invention (I-XVI) and bradykinin (XVII).

The rabbits were anaesthetized by the administration subcutaneously of1.2 g./kg. of urethane.

TABLE [Blood pressure lowering in percent on rabbits (Dose ng./kg)

Compound No. 1 mg. 2 ng. 5 mg. 11g. 20 mg. 50 11g. 100 ng. 200 11g.

The following are the compounds which are used and the results of whichare set forth in the above table:

I=H-A1a-Phe-I1eu'Gly-Leu-Met-NH II:H-Ala-Phe-Val-Gly-Leu-Met-NHIII=H-Ala-Phe-Leu-Gly-Leu-Met-NH IV=H-Asp (NH-Ala-Phe-Leu-Gly-Leu-Met-NH V=H-Glu (NH -Ala-Phe-Ileu-Gly-Leu-Met-NHVI=H-Glu(NH )-A1a-Phe-Leu-G1yLeu-Met-NH VII=H-Asp(NH-Ala-Phe-Ilue-Gly-Leu-Met-NH VIII:H-Gly-Ala-Phe-Ileu-Gly-Leu-Met-NHIX:H-Gly-Ala-Phe-Val-Gly-Leu-Met-NH X:H-Lys-Ala-Phe-Ileu-Gly-Leu-Met-NHXI=H-Lys-Al a-Phe-Val-Gly-Leu-Met-NH XII=H-Lys-Al a-Phe-Leu-Gly-Leu-Met-NI-I XIII: H-Ser-Ala-Phe-Ileu-Gly-Leu-Met-NHXIV=Pyroglu-Ala-Phe-Leu-Gly-Leu-Met-NHXV=H-Sar-Ala-Phe-I1en-Gly-Leu-Met-NHXVI:H-Sar-Ala-Phe-Val-Gly-Leu-Met-NH XVII=Bradykinin The synthesis ofthe new polypeptides of the present invention can be carried out usingmethods commonly used in the synthesis of polypeptides, preferably usingthe method of mixed anhydrides, the azide method or the carbodiimidemethod, or the activated ester method (compare the monograph ofGreenstein and Winitz, Chemistry of the Amino Acids, Wiley & Sons, NewYork, London (1961)).

The amino acid sequence is advantageously built up in small portions.The functional groups which are not B OC-Ala-OH H-Phe-OMe B O C-Ala-Phe-OMe B O C-Ala-Phe-NI-INII:

B O G-Ala-Phe-VaLGly-OMe B O C-AIwPheIVal-Gly-NHNH:

concerned in the reaction are if necessary intermediately blocked bymeans of common protective groups.

The methods of producing the polypeptides of the present invention areclear from the following reaction scheme I-IV in which the followingsymbols are used: H-Gly-OH=glycerine H-L-Ala-OH=L-alanineH-LPhe-OH=L-phenylalanine H-L-Val-OH=L-valine H-L-Leu-OI-I=L-leucineH-L-Ileu-OH:L-isoleucine H-L-Met-OH=L-methionine H-L-Ser-OH=L-serineH-L-Lys-OH=L-lysine IIIHz H-L-Glu-OH: L-glutamine H-L-Sar-OH=L-sarcosineL-Pyroglu-OH=L-pyroglutaminic acid IIIHz H-L-Asp-OH= L'asparagineCbo=carbobenzoxy BOC=carbo-tert.-butoxy MeOCbo:p-methoxycarbobenzoxyPhNO =p-nitrophenyl Me=methyl Furthermore, in the reaction schematicbelow, the letter L used to indicate the configuration has been omitted:

Reaction scheme I Cbo-Val-OH H-GIy-OMe (Ibo-Val-Gly-OMeH-Val-Gly-Ome-HCI B O C-Ala-Phe-Val-Gly-Lou-Met-NHaH-Ala-Phe-Val-Gly-Leu-Met-N H2 Reaction scheme II Cbo-Ilue-OH H-Gly-OMeGbo-Ilue-Gly-OMe BO O-Ala-Phe-N HNH: H-Ilue-G ly-OMe-HCI (compare React.Sch. I) l BOC-Ala-Phellue-Gly-OMe BOC-Ala-Ihe-Ilue-Gly-NHNHZH-Leu-Met-NHz-HC] l (compare React. Sch. 1)

BOO-Ala-Phe-Ilue-Gly-Leu-Met-NH:

NH: MeOGhodllu-AlaPhe-Ilue-Gly-Leu-Met-NH:

N H: H-llu-Ala-Phe-Ilue-Gly-Leu-Met-NHQ Reaction scheme HIBOC-Ala-PheNHNHz H-Val-Gly-Leu-Met-NHz BOC-Ala-Phe-Val-Gly-Leu-Met-NHz BO C BOC-iys-Ala-Phe-Val-Gly-Leu-Met-NHz H-Lys-Ala-Ihe-Val-Gly-LeuMet-NHzReaction scheme IV B O C-Ser-NHNH: H-Ala-Phe-OMe B 0 C-Ser-Ala-Phe-OMe BO C -Scr-Ala- PhcNHNHz H-Ileu- Gly- L eu-Met-NH;

B O C-Ser-Ala-Phe-Ileu-Gly-Leu-Met-Nlh H-Ser-Ala-Phe-Ileu-Gly-Leu-Met-NHz The following examples are given to further illustratethe present invention. The scope of the invention is not, however, meantto be limited to the specific details of the examples:

EXAMPLE 1 (a) BOC-L-Ala-L-Phe-OMe 9.5 g. (50 mmols) of BOC-L-Ala-OH areconverted with 7 cc. (5O mmols) of triethylarnine and 4.8 cc. (50 mmols)of chloroformic acid-ethyl ester in normal manner in tetrahydrofuraneinto the anhydride, and coupled With H-L-Phe-OMe which has been freedfrom 12.9 g (60 mmols) of the hydrochloride and 8.4 cc. of triethylaminein dimethylformamide. After the working up it is recrystallized fromethyl acetate/petroleum ether. The yield is 14.6 g corresponding to 82%of the theoretical. The melting point is 8889 C. [u] =-7.2 (c.=1,glacial acetic acid), -26.9 (c.= 1; chloroform).

(b) B OC-L-Ala-L-Phe-NHNH BOC-L-Ala-L-Phe-NHNH is obtained from BOC-L-Ala-L-Phe-OMe by the action thereon of eight times the amount ofhydrazine hydrate in methanol at room temperature for 48 hours. Thehydrazide is crystallized out, filtered off under suction and Washedwith ethanol and ether. It can be recrystallized from methanol. Theyield is of the theoretical. The melting point is 162-163 C. [a] ='22.6(c.=1; glacial acetic acid).

(0) Cbo-L-Val-Gly-OMe 25.1 g. mmols) of Cbo-L-Val-OH in tetrahydrofuraneare converted into the mixed anhydride with 14 cc. (100 mmols) oftriethylamine and 1 0 cc. 100 mmols) of chloroformic acid-ethyl ester at20 C., and reacted with 15.0 g. mmols) of H-Gly-OMe-HCI and 16.8 cc.(120 mmols) of triethylamine dissolved in dimethylformamide. Afterworking up in the usual manner it is recrystallized from ethyl acetate.The yield is 22.9 g. (70% of the theoretical) the melting point is151152 C. [a] =26.4 (c.=-1; glacial acetate acid).

(d) H-L-Val-Gly-OMe 'HCl H-L-Gly-OMe-HCl is produced by catalytichydrogenation of Cbo-L-Val-Gly-OMe with palladium black in methanol andthe equivalent amount of 1N hydrochloric acid. After concentration ofthe solution it is taken up in Water and shaken with ethyl acetate.Evaporation of the water under vacuum results in the formation of thedesired compound, which is only obtained as an oil. The yield is 80 ofthe theoretical.

(e) BOC-L-Ala-L-Phe-L-Val-Gly-OMe 17.5 g. (50 mmols) ofBOC-L-Ala-PheNHNH are suspended in 66 cc. of a 1.5 N solution ofhydrogen chloride in tetrahydrofurane at -20 C. and reacted with 5.6 g.(61 mmols) of tert.-butylnitrite in 30 cc. of tetrahydrofurane at 20 C.After several shakings, the hydrazide goes into solution. The reactionmixture is diluted with 500 cc. of ethyl acetate and shaken with asodium bicarbonate solution at the lowest possible temperature. Afterdrying over sodium sulfate, the azide solution is reacted with the aminocomponent obtained from 15.5 g. (69 mmols) of H-Val-Gly-OMe-HCI and 9.7cc. (69 mmols) of triethylamine in 100 cc. of dimethylformamide, and thereaction mixture is allowed to stand overnight at 0 C. After distilling01f of the solvent the residue is dissolved in chloroform, shaken withwater, 10% citric acid solution, water, saturated sodium bicarbonatesolution and water, dried and concentrated. The yield afterrecrystallization from ethanol/ethyl acetate/ petroleum ether is 17.3 g.which is equivalent to 68% of the theoretical. The melting point is207-209" C. [a] =35.2 (c.=l; glacial acetic acid).

(f) BOC-L-Ala-L-Phe-L-Val-Gly-NHNH 15.2 g. (30 mmols) ofBOC-L-Ala-L-Phe-L-Val-Gly- OMe are dissolved in 200 cc. of n-butanol andheated to 80 C. with 6.0 cc. (120 mmols) of hydrazine hydrate, theheating being continued for 2 hours. After concentration under vacuum,the hydrazide is recrystallized from ethanol. The yield amounts to 12.2g., equivalent to 80% of the theoretical. The melting point is 218219 C.[a] ;y=-25.3 (c.=1; glacial acetic acid).

(g) BOC-L-Leu-L-Met-OMe 24.9 g. (100 mmols) of BOC-L-Leu-OI-I-H O, 13.8cc. (100 mmols) of triethylamine and cc. (100 mmols) of chloroformicacid-ethyl ester in tetrahydrofurane at 20 C., as well as 23.9 g. (120mmols) of I-I-L-Met- OMe-HCl and 17.0 cc. (120 mmols) of triethylaminein dimethylformamide are converted in normal manner toBOC-L-Leu-L-Met-OMe. After recrystallization from pctroleum ether, 32.5g. of the compound are obtained, the yield amounting to 86% of thetheoretical. The melting point is 97-99- C. [a] =36.5 (c.=1; ethanol).

(h) BOC-L-Len-L-Met-NH 3.8 g. of BOC-L-Leu-L-MetOMe are dissolved in cc.of methanol and this solution is saturated with ammonia at 5 C. Afterstanding for three days at room temperature the solution is concentratedunder vacuum and recrystallized from 70% methanol. The yield is 3.3 g.,corresponding to 90% of the theoretical. The melting point is 154155C.[oc] =-35.l (c.:1; dimethylformamide).

3.6 g. of BOC-L-Leu-L-Met-NH are dissolved in cc. of glacial aceticacid. Hydrogen chloride is conducted therethrough at room temperaturefor 1 hour. Ether is subsequently added to precipitate the compound andthe substance is recrystallized from methanol/petroleum ether. The yieldamounts to 3.0 g., corresponding to 90% of the theoretical. The meltingpoint is 188191 C. [a] =+10.2 (c.=1; water).

(k) BOC-L-Ala-L-Phe-L-Val-Gly-L-Leu-L-Met-NH 2.53 g. (5 mmols) ofBOC-L-Ala-L-Phe-L-ValGly- NHNH are converted to the azide with 0.61 cc.(6 mmols) of tert. butylnitrite in 6.6 cc. of a 1.5 N solution ofhydrogen chloride in tetrahydrofurane at 20 C. As described under (e) itis taken up in ethyl acetate, the solution is Washed and dried. 1.9 g.(6 mmols) of H-L- Leu-L-Met-NH 'Hcl are converted into the free amidewith 0.84 cc. (6 mmols) of triethylamine in dimethylformamide, and thesame is reacted with the azide solution in usual manner. The residueobtained after evaporation of the solvent is washed with water, citricacid solution, water, sodium bicarbonate solution and water. The yieldis 2.73 g., corresponding to 74% of the theoretical. The melting pointis 250 C. (with decomposition). [u] =34.7 (c.=1; glacial acetic acid).

(l) H-L-Ala-L-Phe-L-Val-Gly-L-Leu-Met-NH 1.48 g. (2 mmols) ofBOC-hexapeptide-amide are dissolved in 3 cc. of 90% trifluoroacetic acidat 0 C., and the solution is permitted to stand for 45 minutes at roomtemperature. The reaction product is precipitated with ether. Theprecipitate is dissolved in 50% ethanol, the solution is adjusted to pH910 by the addition of a lithium hydroxide solution, and the ethanol isthen mainly distilled off under vacuum. After standing for awhile at 0C., the residue is filtered off under suction and washed with coldwater. The yield is 0.9 g., corresponding to 75% of the theoretical. Themelting point is 243250 C. [001D =-49.3 (c.=1; trifiuoroacetic acid).

EXAMPLE 2 (a) Cbo-L-Ileu-Gly-OMe Cbo-L-Ileu-Gly-OMe is obtained fromCbo-L-lleu-OH and H-Gly-OMe by the anhydride method and recrystallizedfrom ethyl acetate. The yield corresponds to of the theoretical. Themelting point is 127-130 C. [a] =-26.6 (c.=2; glacial acetic acid).

Literature citation: Determann and Wieland, Makromolekulare Chemie, 44(1961), p. 312.

H-L-Ileu-Gly-OMe-HCI is obtained from the carbobenzoxy compound byhydrogenation with palladium black as catalyst. After recrystallizationfrom methanol/ ether the yield amounts to 87% of the theoretical. Themelting point is 18l182 C. [a] =+l6.3 (c.=1; glacial acetic acid).

Literature citation: Determann and Wieland, Makromolekulare Chemie 44(1961), p. 312.

(c) BOC-L-Ala-L-Phe-L-Ileu-Gly-OMe 8.9 g. (25 mmols) ofBOC-L-Ala-L-Phe-NHNH are suspended in 33 cc. of a 1.5 N solution ofhydrogen chloride in tetrahydrofurane at 20 C., and reacted with 2.8 g.(27 mmols) of tert.-butylnitrite dissolved in 15 cc. oftetrahydrofurane, at a temperature of 20 C. After some shaking, thehydrazide goes into solution. The reaction mixture is diluted with 250cc. of glacial acetic acid and shaken at the lowest possible temperaturewith a sodium bicarbonate solution. After drying over sodium sulfate,the azide solution is reacted with the mixture of 7.1 g. (30 mmols) ofH-L-lleu-Gly-OMe and 4.2 cc. (30 mmols) of triethylamine in 50 cc. ofdimethylformamide, and the reaction mass is permitted to stand overnightat 0 C. After distilling off of the solvent, the residue is dissolved inchloroform, shaken with water, 10% citric acid, water, saturated sodiumbicarbonate solution and water, then dried, concentrated, andrecrystallized from ethanol/ petroleum ether. The yield is 9.2 g. (70%of the theoretical). The melting point is 201-204 C. [a] -=34.5 (c.=1;glacial acetic acid).

The same compound can be obtained in the following manner:BOC-L-Ala-L-Phe-OMe (note Example 1(a)) is saponified in ethanol withthe equivalent amount of 1 N KOH in 40 minutes, most of the ethanol isdistilled off under vacuum, and after acidification, the residue isextracted with ethyl acetate. After washing to neutral and drying, theBOC-LAla-L-Phe-OH is isolated as a vitreous mass. The yield correspondsto 90% of the theoretical. [a] =+5.l (c.:1; glacial acetic acid).

3.4 g. (10 mmols) of BOC-L-Ala-L-Phe-OH are dissolved in 10 cc. oftetrahydrofurane and reacted at 10 C. with a solution ofH-L-Ileu-Gly-OMe which has been produced from 3.5 g. of thehydrochloride and 2.1 cc. of triethylamine in 20 cc. ofdimethylformamide. After the addition of 2.5 g. (12 mmols) ofN,N-dicyclohexylcarbodiimide in 5 cc. of tetrahydrofurane, the reactionmixture is stored for 24 hours at 0 C. and subjected to suctionfiltration to separate the N,N-dicyclohexylurea. It is then furtherworked up as described above. The yield is 4.5 g., corresponding to ofthe theoretical.

(d) BOC-L-Ala-L-Phe-L-Ileu-Gly-NHNH butylnitrite in 15 cc. of a 1.5 Nsolution of hydrogen chloride in tetrahydrofurane. Several drops ofwater have to be added in order to have the azide go completely intosolution. After dilution with 100 cc. of ethyl acetate, the reactionmass is shaken, dried and coupled with a solution of 4.8 g. 15 mmols) ofH-L-Leu-L-Met- NH -'I-ICl-H and 2.1 cc. (15 mmols) of triethylamine indimethylformamide. The reaction mixture is stored for two days at 0 C.,and the solvent is then evaporated under vacuum. The residue is washedWith Water, citric acid solution, water, saturated sodium bicarbonate,solution, extremely thoroughly, and then dried. The yield is 5.8 g.corresponding to 77% of the theoretical. The melting point is 248250 C.[a] =29.7 (c.=1; dimethylformamide.

( f H-L-Ala-L-Phe-Ileu-Gly-L-Leu-L-Met-NH 4.5 g. (6 mmols) ofBOC-hexapeptide-amide are mixed with cc. of 90% trifiuoroacetic acid at0 C., and the reaction mixture is allowed to stand for 1 hour at thistemperature. After precipitation by means of ether, the reaction mixtureis subjected to suction filtration and the filter residue is dissolvedin 10 cc. of 50% ethanol. The pH is adjusted to between 9-10 by theaddition of a lithium hydroxide solution, and then the ethanol is to agreater extent distilled oil. To complete the precipitation, thereaction mixture is permitted to stand for some time at 0 C. The yieldis 2.7 g. corresponding to 70% of the theoretical. The melting point is241244 C. [a] =60.0 (c.=0.5; trifluoroacetic acid).

0.65 g. (1.5 mmols) of MeOCbo-L-Gly(NH )-OPhNO are dissolved in 2 cc. ofethyl acetate and added to a solution of 1.04 g. (1.6 mmols) ofH-L-Ala-L-Phe-L- Ileu-Gly-L-Leu-L-Met-NH in dimethylformamide. Thereaction mixture is stored for 4 days at 40 C. After concentration undervacuum it is cooked with ethyl acetate and the residue is treated with10% citric acid solution and water. The yield is 1.06 g., correspondingto 75% of the theoretical. The melting point is 267-269 C. (withdecomposition) 892 mg. (1 mmols) of MeOCbO-heptapeptide-amide are mixedwith 0.2 cc. of anisole and 1.3 cc. of trifluoroacetic acid andpermitted to stand for 1 hour at room temperature. After precipitationby means of ether, the reaction mixture is dried under vacuum overpotassium hydroxide. It is then dissolved in 50% ethanol, the pH isadjusted to a value of 9 by means of a lithium hydroxide solution, andthe ethanol is distilled off under vacuum. After standing for some timeat 0 C., the reaction mixture is subjected to suction filtration andthen dried over phosphorus pentoxide. The yield is 540 mg.,corresponding to 70% of the theoretical. The melting point is 245- 258C. (with decomposition). [a] =-68.5 (c.=0.5; trifiuoroacetic acid).

Analogously to Example 2, instead of usingp-methoxycarbobenzoxy-L-glutamine-pnitrophenyl ester, it is possible touse p-methoxycarbobenzoxy-L-asparagine-p-nitrophenyl ester to produce:

The melting point is 215-240 C. [a] =22.2 (c.=0.5; dimethylforrnamide.

correspondingly, instead of using carbobenzoxy-L-isoleucine, by the useof carbobenzoxy-L-leucine, it is possible to produce:

NHz H-L(ElwLAlaL-Phe-L-Leu-G1y-L-Leu-L-Met-NH1 which melts at 214-220 C.(with decomposition).

10 [a] =62.8 (c.=0; trifluoroacetic acid) it is also possible toproduce:

NH: H-LAisp-L-Ala-L-Phe-LLeu-Glx-L-Leu-L-Met-NHz which melts at 233-243C. (with decomposition). [a] =-77.2 (c.=0.5; trifluoroacetic acid).

EXAMPLE 3 (a) H-L-Ala-L-Phe-OMe-HCI 69.5 g. of BOC-L-AIa-L-Phe-OMearedissolved in cc. of glacial acetic acid. Hydrogen chloride is thenconducted therethrough for 30 minutes. After an additional 30 minutesprecipitation is accomplished by means of ether, and the precipitatedproduct is recrystallized from methanol/ether. The yield is 45.9 g.,corresponding to 80% of the theoretical. The melting point is 154-156 C.[041 58.5 (c.=1, glacial acetic acid).

(b) BOC-Gly-L-Ala-L-Phe-OMe 17.5 g. of BOC-Gly-OH in cc. ofdimethylformamide are converted to the mixed anhydn'de at -15 C. with14.0 cc. of triethylamine and 10.0 cc. of chloroformic acid-ethyl ester,and reacted with a solution of 31.5 g. of H-L-AlaL-Phe-OMe'HCl and 15.4cc. of triethylamine in 150 cc. of dimethylformamide. After working upin the usual manner there is obtained 31.5 g., which corresponds to 77%of the theoretical. The melting point is 106108% C. [oc] 24.6 (c.=1, inethanol).

(0) BOC-Gly-L-Ala-L-Phe-NHNH The compound is obtained from the methylester by reaction with four times the amount of hydrazine hydrate inmethanol. The yield is 89% of the theoretical. The melting point is177-183 C. [ab 2.7.7 (c.=l, glacial acetic acid).

(d) BOCL-Ileu-Gly-L-Leu-L-Met-NH 22.0 g. (84% of the theoretical) ofBOC-L-Ileu-Gly-L-Leu-L-Met-NH are obtained from 11.6 g. of BOC-L-Ileu-OHand 21.3 g. of H-GIy-L-Leu-LMet-NH -HCI by means of the anhydride method(cloroforrnic acid-ethyl ester, triethylamine in dimethylformamide at-15 C.). The melting point of the compound is 207-209" C. [a] =33.2(c.'=1, glacial acetic acid).

21.2 g. of the obtained BOG-compound are dissolved in 150 cc. of glacialacetic acid. The protective group is split oh? by conducting hydrogenchloride therethrough for /2 hour. After precipitation with ether, thecompound is recrystallized from methanol/ether. The yield is 14.0 g.,corresponding to 75 of the theoretical, of

The compound melts at -200 C. (with decomposition). [a] =-27.l (c=.1,methanol).

(f) BOC-Gly-L-Ala-L-Phe-L-Ileu Gly-L-Leu-L-Met-NH 1.22 .g. ofBOC-Gly-L-Ala-L-Phe-NHNH are suspended in 3.6 cc. of a 2.2 N solution ofhydrogen chloride in tetrahydrofurane at -20 C., and converted with 0.37cc. of tert.-butylnitrite into the azide. After addition of 40 cc. ofcold ethyl acetate it is Washed Wit-h a saturated solution of sodiumbicarbonate and dried over sodium sulfate. This azide solution isreacted with a mixture of 1.4 g. of H-L-Ileu-Gly-L-Leu-L-Mt-NH -HCl and0.42 cc. of triethylamine in 15 cc. of dimethylformarnide. Afterconcentration, it is washed with 10% citric acid solution, water, sodiumbicarbonate solution and water, and then dried. The yield is 2.16 g.,corresponding to 89% of the theoretical, of

BOC-Gly-L-Ala-L-Phe-Ldleu-Gly-L-Leu-L-Met-NH 11 which melts at 252225 C.[a] =27.0 (c.:l, dimethylformamide)H-Gly-L-Ala-L-Phe-L-Ileu-Gly-L-Leu-L-Met-NH HCl 1.5H O is obtained in ayield of 1.6 g. (88% of the theoretical) from 2.0 g. of the BOG-compoundby splitting off of the protective blocking group by means by hydrogenchloride in glacial acetic acid as previously described. The meltingpoint is 240-260 C.

(c.=1, glacial acetic acid).

The corresponding valine derivative is obtained in analogous manner fromBOC-Gly-L-Ala-L-Phe-NHNH; and H-L-Val-Gly-L-Leu-L-Met-NH By splittingoff of the protective groups the resultingheptapeptide-amidehydrochloride is obtained which is then converted intothe free base by means of lithium hydroxide in ethanol/ water. The freebase is M.P. 225-229 C. [a] =52.Z (c:1, trifiuoroacetic acid).

The necesary tetrapeptide-amide which is needed for this synthesis canalso be obtained from BOC-L-Val-Gly-NHNH and H-L-Leu-L-Met-NHH-Sar-L-Ala-L-Phe-L-Ileu-Gly-L-Leu-L-Met-NH -HC1,

M.P. 242244 C. [a] =45.9 (c.:1, glacial acetic acid), and

H-Sar-L-Ala-L-Phe-L-Val-Gly-L-Leu-L-Met-NH HCl M.P. 248249 C. [a] =43.2(c:1, glacial acetic acid), are obtained in analogous manner fromBOC-Sar-L-Ala-L-Phe-NHNH and the corresponding tetrapeptide-amide.

EXAMPLE 4 (a) BOC-L-Lys(BOC)-L-AlaLPhe-L-Val-Gly-L-Leu-L- Met-NH Thecompound obtained according to Example 1(k) is subjected to splittingoil? of the BOG-group by means of trifluoroacetic acid, and the reactionproduct by dissolution in ethanol, addition of an equivalent amount oflithium hydroxide and precipitation with water is converted into thefree amine. 6.3 g. thereof is dissolved in dimethylformamide and mixedwith a solution of 4.7 g. of Di-BOC-L- Lysine-OPhNO in ethyl acetate andmaintained therein for 3 days at 40 C. The resulting precipitatedreaction product is washed with ether. The yield is 7.5 g. The meltingpoint is 237239 C. [c] =37.3 (c=0.5 glacial acetic acid).

(b) H-L-Lys-L-Ala-L-Phe-L-Val-Gly-L-Leu-L-Met-NHH-L-Lys-L-Ala-L-Phe-L-Val-Gly-L-Leu-L-Met-NH can be obtained from theBOC-compound by splitting off of the protective group with hydrogenchloride in glacial acetic acid. It is subjected to chromatography on acarboxy methyl cellulose column for purification (a-mmonium acetategradient, pH 5.5; 0.001 to 0.2 molar).

In analogous manner it is possible to obtain: H-Lys-Ala-Phe-Ileu-Gly-Leu-Met-NH and H-Lys-Ala-Phe-Leu Gly- Leu-Met-NH EXAMPLE 5L-Pyroglu-L-Ala-L-Phe-L-Leu-Gly-L-Leu-L-Met-NH 0.45 g. ofMeOCbo-L-Pyroglu-OPhNO are reacted as described in Example 4(a) with0.78 g. of the hexapeptide H-L-Ala-L-Phe-L-Leu-Gly-L-Leu-L-Met-NH(obtained analogously to the Val-compound (note Example 1)). The yieldis 0.55 g. The melting point is 238245 C. The protective group is splitoff from thiscompound by means of trifiuoroacetic acid in the presenceof anisole. For purification purposes the compound is rubbed severaltimes with hot ethanol and then filtered off under suction. The meltingpoint is 279-281 C. (with decomposition). [a] =--54.7 (c.=0.5, glacialacetic acid).

EXAMPLE 6 This example is carried out in accordance with Schema TV.

(a) BOC-L-Ser-L-Ala-L-Phe-NHNI-l BOC-L-Ser-NHNH is converted into theazide in normal manner by means of tert.-butylnitrite, and then reactedwith H-L-Ala-L-Phe-OMe. The thus obtained tripeptide-methyl ester isconverted into the hydrazide by means of hydrazine hydrate.

(b) BOC-L-Ser-L-Ala-L-Phe-L-Ileu-Gly-L-Leu-L-Met- The above compound isobtained by means of the azide method in normal manner as shown inExample 6a from BOC-L-Ser-L-Ala-L-Phe-NHNH and H-L-Ileu-Gly-L-Leu-L-Met-NH (c) H-L-Ser-L-Ala-L-Phe-L-Ileu-Gly-L-Leu-L-Met-NH The abovecompound is obtained from the BOC-co-rnpound by splitting off of theprotective group with trifluoroacetic acid and normal purificationprocedures.

Without further analysis, the foregoing will so fully reveal the gist ofthe present invention that others can by applying current knowledgereadily adapt it for various applications without omitting featuresthat, from the standpoint of prior art, fairly constitute essentialcharacteristics of the generic or specific aspects of this inventionand, therefore, such adaptations should and are intended to becomprehended within the meaning and range of equivalence of thefollowing claims.

What is claimed as new and desired to be secured by Letters Patent is:

1. Polypeptide of the formula:

R-L-alanyl-L-phenylalanyl-L-leucyl-glycyl-L-leucyl- L-methioninamidewherein R is selected from the group consisting of hydrogen and naturalL-a-amino acid radicals selected from the group consisting of radicalsof glycine, alanine, valine, leucine, isoleucine, asparagine, glutamine,serine, threonine, cysteine, methionine, phenylanalanine, tyrosine,proline, oxyproline, lysine, histidine, arginine, ornithine, norleucine,norvaline, zx-fiflllllOblltYflC acid, Dt,'Y-diamiI10- butyric acid,pyroglutamic acid, sarcosine, N-methylvaline, N-methylleucine, andtryptophane.

2. Polypeptide of the formula:

R-L-alanyl-L-phenylalanyl-L-valyl glycyl-L-leucyl-L- methioniamidewherein R is selected from the group consisting of hydrogen and naturalL-ot-amino acid radicals selected from the group consisting of theradicals of glycine, alanine, valine, leucine, isoleucine, asparagine,glutamine, serine, threonine, cysteine, methionine, phenylanalanine,tyrosine, proline, oxyproline, lysine, histidine, arginine, ornithine,norleucine, norvaline, m-aminobutyric acid, a,'y-diaminobutyric acid,pyroglutamic acid, sarcosine, N-methylvaline, N-methylleucine andtryptophane.

3. L-alanyl-L-phenylalanyl-L-leucyl-glycyl-L leucyl-L- methioninamide.

4. Glycyl-L-alanyl-L phenylalanyl-L-leucyl-glycyl-L-leucyl-L-methioninamide.

5. Glycyl-L-alanyl L-phenylalanyl L-valyl-glycyl-L-leucyl-L-methioninamide.

6. L-lysyl-L-alanyl-Lphenylalanyl-L leucyl-glycyl-L-leucyl-methioninamide.

1. POLYPEPTIDE OF THE FORMULA:
 2. POLYPEPTIDE OF THE FORMULA: 13.L-LYSYL-ALANYL-L-PHENYLALANYL - L -ISOLEUCYL-GLYCYL-L-LEUCY-METHIONINAMIDE.